Interpretive Summary: Root-knot nematodes (RKNs) (Meloidogyne sp.) infect a wide range of plants, including grapes (Vitis vinifera L.). The impact of RKNs on grape growth and production are especially severe for grapes grown on warm sandy soils and more than half of the California vineyard acreage, which accounts for 90% of US grape production, is affected. Nematode management through fumigation, particularly by using methyl bromide, can be useful in mitigating infestation. However, the use of chemicals is not only costly but also environmentally harmful. The most cost-effective solution to control RKNs in vineyards is to use nematode resistant rootstocks, but breeding for nematode-resistant rootstocks is a long and laborious process. Furthermore, the occurrence of multiple RKN species and the constant emergence of new virulent populations in vineyards make the conventional breeding for nematode resistant rootstocks even more challenging. In this study we evaluated and demonsatrted the feasibility of generating RKN resistance in transgenic grape hairy roots via a biotech approach, RNAi. The knowledge obtained from this study will help develop an alternative solution to addressing the devastating RKN problem in vineyards in the future. The study also provided some insights into how to optimize various components of the RNAi technology for enhancing its effectiveness against RKNs in transgenic grapes.

Technical Abstract:
Root-knot nematodes (RKNs) infect many annual and perennial crops and are the most devastating soil-born pests in vineyards. To develop a biotech-based solution for controlling RKNs in grapes, we evaluated the efficacy of plant-derived RNA interference (RNAi) silencing of a conserved RKN effector gene, 16D10, for nematode resistance in transgenic grape hairy roots. Two hairpin-based silencing constructs, containing a stem sequence of 42 bp (pART27-42) or 271 bp (pART27-271) of the 16D10 gene, were compared for nematode resistance and small interfering RNA (siRNA) production. Transgenic hairy root lines expressing either of the two constructs showed less susceptibility to nematode infection compared with control and the pART27-42 lines was more effective than the pART27-271 lines in suppressing nematode infection. Small RNA libraries from selected pART27-42 and pART27-271 hairy root lines were sequenced using an Illumina sequencing technology. The pART27-42 lines produced hundred times more 16D10-specific siRNAs than the pART27-271 lines. On average the 16D10 siRNAs had higher GC content than the 16D10 stem sequences in the RNAi constructs, supporting that plant dicer-like enzymes prefer GC-rich sequences as substrates for siRNA production. The stems of the 16D10 RNAi constructs were not equally processed into siRNAs. Several hotspots of siRNA production were found in similar positions in pART27-42 and pART27-271. Interestingly, stem sequences near at the loop terminus produced more siRNAs than those at the stem base. Furthermore, the relative abundance of guide and passenger siRNAs from putative siRNA duplexes largely correlated with their 5’ end thermodynamic strength. This study demonstrated the feasibility of using plant-derived RNAi of nematode effector genes for the generation of novel nematode resistance in grapes and revealed several molecular characteristics of transgene siRNAs important for optimizing plant RNAi constructs.